Macrophage cells of the immune system concentrate copper (Cu) into phagosomes to intensify microbial killing, while microbes counteract by upregulating Cu resistance pathways. There is an unmet opportunity to create innovative antimicrobial agents that manipulate Cu along this host/pathogen interface, and there remain significant gaps in understanding the mechanisms of Cu in immunity and microbial toxicity. The long-term goal is to develop chemical tools to manipulate biological metal ion location, speciation, and reactivity for potential therapeutic benefit. The overall objective of the current application is to use triggerable metal-binding agents, called prochelators, to manipulate Cu in innate immune cells to kill infecting microbes. The central hypothesis is that small molecules that can be triggered to mobilize Cu selectively in response to infection can boost the immune system's use of bactericidal Cu, evade the Cu resistance pathways of the pathogen, and avoid disrupting the overall metal status of the host. This hypothesis is formulated based on preliminary in vitro data from the applicant's laboratory showing that select prochelators are triggered by reactants associated with activated macrophages to convert non-toxic prodrugs into potent Cu-dependent fungicides. The hypothesis will be further tested in the fungal pathogen Cryptococcus neoformans by addressing three specific aims: 1) Identify chelator/prochelator pairs that enhance Cu-stimulated microbial killing but avoid mammalian cell toxicity; 2) Delineate mode of action of Cu-dependent microbial killing; and 3) Develop multiresponsive fluorescent probes to visualize metal redistribution in response to macrophage activation. Under the first aim, small molecules will be assayed for Cu-dependent microbicidal activity and prochelator versions will be synthesized and assayed for mammalian cell viability. Promising compounds will be tested for infection clearance by macrophages and characterized with respect to prochelator properties. Preliminary results demonstrate feasibility of these assays and prochelator synthesis/characterization strategies by the applicant. The second aim benefits from an established collaboration to combine biochemical, genetic, and analytical testing to elucidate how a fungal pathogen responds, adapts, and succumbs to Cu delivered by a potential therapeutic agent. The third aim builds on the applicant's experience in designing fluorescent probes to create fluorescent prochelators capable of sensing metal ions in response to the changing chemical environment induced by macrophage activation. The overall approach is innovative because it exploits the unique chemical milieu created by the host in response to infection to mobilize endogenous Cu to exacerbate microbial killing. The proposed research is significant because it represents the first step in developing broad-spectrum antimicrobial agents based on Cu biology while elucidating mechanisms of Cu-induced microbial toxicity.